PF234 SIGNIFICANT REDUCTION OF REGULATORY INNATE LYMPHOID CELLS (ILCREGS, CD45+LIN‐CD127+IL‐10+) AND CD45+LIN‐CD127+IL‐10‐ CELLS IN PATIENTS WITH ACUTE MYELOID LEUKEMIA DETECTED BY FLOW CYTOMETRY

Background: A new regulatory subpopulation of ILCs, ILCregs has been identified in mouse and human intestines. ILCregs share characteristics with both innate lymphoid cells and regulatory cells. The significance of CD45+Lin‐CD127+IL‐10+ ILCregs in patients with AML remains unclear. Also, the CD45+Lin‐CD127+IL‐10‐ subset population has not been classified and its significance remains unknown. Aims: To explored the difference of CD45+Lin‐CD127+IL‐10+ ILCregs population and the CD45+Lin‐CD127+IL‐10‐ subset population by flow cytometry between the normal donors and patients with AML. Methods: Flow cytometry assay: Bone marrow(BM) cells from 12 normal donors and 42 patients newly diagnosed with AML were collected and cultured in media containing Bredfeldin A for 4 hr at 37°C. Cells were harvested and stained with surface markers CD45‐FITC, Lin‐APC,CD127‐PE‐Cy7 for 15 minutes. Red blood cells were lysed with lysing buffer. Cells were then fixed and permeablized by Intracellular Fixation & Permeablization buffer set (eBioscience) after surface marker staining, followed by application of anti‐IL‐10‐PE antibody (JES5–16E3) staining intracellular IL‐10 before analyzed through flow cytometry (FACS Aria III, BD). Whole cell populations with SSC vs FSC were selected and gated on the CD45+Lin‐ population with further gating of CD127+IL‐10+ subset and CD127+IL‐10‐ subset. At least a total of 1 million cells were collected for analysis. ILCregs cells were defined as CD45+Lin‐CD127+IL‐10+ population. Results: Using the 4 colors monoclonal antibody combination, we were able to detect the ILCregs defined as CD45+Lin‐CD127+IL‐10+ in the BM from both normal donor and AML patients. The frequency of ILCregs in normal donors were 0.9958 ± 1.0730% and 2.2941 ± 2.3533% in whole BM cells from all events gating and CD45+Lin‐ cell population gating, respectively. In addition, the frequency of ILCregs in AML patients were 0.2434 ± 0.5344% and 0.8924 ± 1.3791% in whole BM cells from all events gating and CD45+Lin‐ cell population gating, respectively. In comparison with the normal donors, the frequency of ILCregs cells in AML patients were significantly decreased for both whole BM cells gating and CD45+Lin‐ cell population gating (both P  < 0.01). The frequency of CD45+Lin‐CD127+IL‐10‐ subset in normal donors were 4.0869 ± 6.7701% and 8.2445 ± 11.1016% in whole BM cells from all events gating and CD45+Lin‐ cell population gating, respectively. In addition, the frequency of CD45+Lin‐CD127+IL‐10‐ cell frequency in AML patients were 0.2769 ± 0.2526% and 1.2053 ± 1.7382% in whole BM cells from all events gating and CD45+Lin‐ cell population gating, respectively. In comparison with the normal donors, the frequency of CD45+Lin‐CD127+IL‐10‐ cells in AML patients were also significantly decreased for both whole BM cells gating and CD45+Lin‐ cell population gating (both P  < 0.01). Summary/Conclusion: By using the combination of surface and IL‐10 for intracellular staining analyzed by flow cytometry, we were able to detect ILCregs in BM from both normal donors and patients with AML. The frequency of the ILCregs and CD45+Lin‐CD127+IL‐10‐ subset populations were both significantly decreased in the patients with AML comparing to normal donors. Further studies need to be done to explore the significance of ILCregs and CD45+Lin‐CD127+IL‐10‐ subset populations in AML patients.